Phosphate tightly associated with protein has been known since the late nineteenth century. Since then, a variety of covalent linkages of phosphate to proteins have been found. The most common involve esterification of phosphate to serine, threonine, and tyrosine with smaller amounts being linked to lysine, arginine, histidine, aspartic acid, glutamic acid, and cysteine. The occurrence of phosphorylated proteins implies the existence of one or more protein kinases capable of phosphorylating amino acid residues on proteins, and also of protein phosphatases capable of hydrolyzing phosphorylated amino acid residues on proteins.
Protein kinases play critical roles in the regulation of biochemical and morphological changes associated with cellular growth and division (D""Urso, G. et al. (1990) Science 250: 786-791; Birchmeier. C. et al. (1993) Bioessays 15: 185-189). They serve as growth factor receptors and signal transducers and have been implicated in cellular transformation and malignancy (Hunter, T. et al. (1992) Cell 70: 375-387; Posada, J. et al. (1992) Mol. Biol. Cell 3: 583-592; Hunter, T. et al. (1994) Cell 79: 573-582). For example, protein kinases have been shown to participate in the transmission of signals from growth-factor receptors (Sturgill, T. W. et al. (1988) Nature 344: 715-718; Gomez, N. et al. (1991) Nature 353: 170-173), control of entry of cells into mitosis (Nurse, P. (1990) Nature 344: 503-508; Maller, J. L. (1991) Curr. Opin. Cell Biol. 3: 269-275) and regulation of actin bundling (Husain-Chishti, A. et al. (1988) Nature 334: 718-721). Protein kinases can be divided into two main groups based on either amino acid sequence similarity or specificity for either serine/threonine or tyrosine residues. A small number of dual-specificity kinases are structurally like the serine/threonine-specific group. Within the broad classification, kinases can be further sub-divided into families whose members share a higher degree of catalytic domain amino acid sequence identity and also have similar biochemical properties. Most protein kinase family members also share structural features outside the kinase domain that reflect their particular cellular roles. These include regulatory domains that control kinase activity or interaction with other proteins (Hanks, S. K. et al. (1988) Science 241: 42-52).
The present invention is based, in part, on the discovery of novel protein kinase family members, referred to herein as xe2x80x9c2504, 15977, and 14760xe2x80x9d. The nucleotide sequence of a cDNA encoding 2504 is shown in SEQ ID NO:1, and the amino acid sequence of a 2504 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:3. The nucleotide sequence of a cDNA encoding 15977 is shown in SEQ ID NO:4, and the amino acid sequence of a 15977 polypeptide is shown in SEQ ID NO:5. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:6. The nucleotide sequence of a cDNA encoding 14760 is shown in SEQ ID NO:7, and the amino acid sequence of a 14760 polypeptide is shown in SEQ ID NO:8. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:9.
Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 2504, 15977, or 14760 protein or polypeptide, e.g., a biologically active portion of the 2504, 15977, or 14760 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8. In other embodiments, the invention provides isolated 2504, 15977, or 14760 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 1843. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 1843. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 1843. wherein the nucleic acid encodes a full length 2504, 15977, or 14760 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 2504, 15977, or 14760 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 2504, 15977, or 14760 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 2504, 15977, or 14760 nucleic acid molecules and polypeptides.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 2504, 15977, or 14760-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 2504, 15977, or 14760 encoding nucleic acid molecule are provided.
In another aspect, the invention features, 2504, 15977, or 14760 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 2504, 15977, or 14760 mediated or related disorders. In another embodiment, the invention provides 2504, 15977, or 14760 polypeptides having a 2504, 15977, or 14760 activity. Preferred polypeptides are 2504, 15977, or 14760 proteins including at least one protein kinase domain, e.g. a serine/threonine kinase domain, and, preferably, having a 2504, 15977, or 14760 activity, e.g., a 2504, 15977, or 14760 activity as described herein.
In other embodiments, the invention provides 2504, 15977, or 14760 polypeptides, e.g., a 2504, 15977, or 14760 polypeptide having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8; the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number 1843; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 1843, wherein the nucleic acid encodes a full length 2504, 15977, or 14760 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 2504, 15977, or 14760 nucleic acid molecule described herein.
In a related aspect, the invention provides 2504, 15977, or 14760 polypeptides or fragments operatively linked to non-2504, 15977, or 14760 polypeptides to form fusion proteins.
In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 2504, 15977, or 14760 polypeptides.
In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 2504, 15977, or 14760 polypeptides or nucleic acids.
In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) the proliferation, survival, and/or differentiation of a cell, e.g., a 2504-, 15977-, or a 14760-expressing cell, e.g., a neural cell (e.g., a brain or glial cell), a cardiovascular cell (e.g., a heart or blood vessel cell, e.g., a smooth muscle cell), a liver cell, a hematopoietic cell (e.g., a bone marrow cell such as a glycophorin-positive cell). The method includes contacting the cell with an agent (e.g., a screened compound) that modulates the activity or expression of a 2504-, 15977-, or a 14760 polypeptide or nucleic acid, in an amount effective to modulate the proliferation and/or differentiation of the cell.
In a preferred embodiment, the 2504-, 15977-, or a 14760 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2, 5 or 8. In other embodiments, the 2504-, 15977-, or a 14760 polypeptide is a fragment of at least 15, 20, 50, 100, 150, or more contiguous amino acids of SEQ ID NO:2, 5 or 8.
In a preferred embodiment, the 2504-, 15977-, or a 14760 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1, 3, 4, 6, 7, or 9. In other embodiments, the 2504-, 15977-, or a 14760 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more contiguous nucleotides of SEQ ID NO:1, 3, 4, 6, 7, or 9.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein kinase activity.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 2504-, 15977-, or a 14760 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 2504-, 15977-, or a 14760 nucleic acid, or any combination thereof.
In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.
In a preferred embodiment, the cell, e.g., the 2504-, 15977-, or a 14760-expressing cell, is a neural cell (e.g., a neuronal or glial cell), a cardiovascular cell (e.g., a heart or blood vessel cell, e.g., a smooth muscle cell), a liver cell, a hematopoietic cell, e.g., a myeloid, lymphoid or erythroid cell, or a precursor cell thereof. Examples of such cells include myelocytic cells (polymorphonuclear cells), erythrocytic cells, lymphocytes, monocytes, reticular cells, plasma cells and megakaryocytes, as well as stem cells for the different lineages, and precursors for the committed progenitor cells, for example, precursors of blood cells (e.g., red blood cells, such as erythroblasts), macrophages (monoblasts), platelets (megakaryocytes), polymorphonuclear leucocytes (myeloblasts), and lymphocytes (lymphoblasts).
In a preferred embodiment, the cell, e.g., the 14760-expressing cell, is a bone marrow erythroid cell, e.g., an erythroid progenitor (e.g., a glycophorin A expressing cell) or a differentiated cell, e.g., an erythrocyte or a megakaryocyte.
In a preferred embodiment, the cell, e.g., the 2504-, 15977-, or a 14760-expressing cell, is further contacted with a protein, e.g., a cytokine or a hormone. Exemplary proteins include, but are not limited to, G-CSF, GM-CSF, stem cell factor, interleukin-3 (IL-3), IL-4, Flt-3 ligand, thrombopoietin, and erythropoietin. Most preferably, the protein is erythropoietin. The protein contacting step can occur before, at the same time, or after the agent is contacted. The protein contacting step can be effected in vitro or ex vivo. For example, the cell, e.g., the 14760-expressing cell is obtained from a subject, e.g., a patient, and contacted with the protein ex vivo. The treated cell can be re-introduced into the subject. Alternatively, the protein contacting step can occur in vivo.
In a preferred embodiment, the agent and the 2504-, 15977-, or a 14760-polypeptide or nucleic acid are contacted in vitro or ex vivo.
In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. Preferably, the subject is a human, e.g., a patient with an immune, cardiovascular, proliferative, or liver disorder. In other embodiments, the subject is a non-human animal, e.g., an experimental animal.
The contacting step(s) can be repeated.
In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation of the cell, e.g., the 2504-, 15977-, or a 14760-expressing cell. Such agents can be used to treat or prevent cancers, e.g., leukemic cancers such as erythroid leukemias, or carcinomas,
In preferred embodiments, the methods involve treatment or prevention of disorder related to aberrant activity or expression of the 2504, 15977, or 14760 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular proliferation or differentiation, neural disorders, immune disorders, cardiovascular disorders, liver, skin, and skeletal muscle disorders, among others. The method includes administering to the subject an effective amount of an agent that modulates the activity or expression of a 2504, 15977, and 14760 polypeptide or nucleic acid such that the disorder is ameliorated or prevented.
In a preferred embodiment, the 2504, 15977, and 14760 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2, 5 or 8. In other embodiments, the 2504, 15977, and 14760 polypeptide is a fragment of at least 15, 20, 50, 100, 150, or more contiguous amino acids of SEQ ID NO:2, 5 or 8.
In a preferred embodiment, the 2504, 15977, and 14760 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1, 3, 4, 6, 7 or 9. In other embodiments, the 2504-, 15977-, or a 14760 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more contiguous nucleotides of SEQ ID NO:1, 3, 4, 6, 7 or 9.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein kinase activity.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 2504, 15977, and 14760 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 2504, 15977, and 14760 nucleic acid, or any combination thereof.
In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.
In a preferred embodiment, the subject is a human, e.g., a patient with a disorder described herein. In other embodiments, the subject is a non-human animal, e.g., an experimental animal.
In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation of a cell, e.g., a 2504, 15977, and 14760-expressing cell, e.g., a hematopoietic cell, in the subject. Such agents can be used to treat or prevent cancers, e.g., leukemic cancers such as erythroid leukemias, or carcinomas.
In a preferred embodiment, the disorder is an immune disorder, a cardiovascular disorder, a neural disorder, a liver disorder, among others.
The administration of the agent and/or protein can be repeated.
The invention also provides assays for determining the activity of or the presence or absence of 2504, 15977, or 14760 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In further aspect the invention provides assays for determining the presence or absence of a genetic alteration in a 2504, 15977, or 14760 polypeptide or nucleic acid molecule, including for disease diagnosis.
The invention also features a method of diagnosing, or staging, a disorder, e.g., a disorder as described herein, in a subject. The method includes evaluating the expression or activity of a 2504, 15977, and 14760 nucleic acid, or a 2504, 15977, and 14760 polypeptide, such that, a difference in the level of 2504, 15977, and 14760 nucleic acid, or 2504, 15977, and 14760 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of the disorder, or a stage in the disorder.
In a preferred embodiment, the subject is a human.
In a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample or biopsy, is obtained from the subject.
In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 2504, 15977, and 14760 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 2504, 15977, and 14760 nucleic acid or polypeptide.
In still another aspect, the invention features a method for evaluating the efficacy of a treatment of a disorder (e.g., a disorder as described herein), in a subject. The method includes treating a subject with a protocol under evaluation; assessing the expression of a 2504, 15977, or 14760 nucleic acid, or 2504, 15977, or 14760 polypeptide, such that a change in the level of the 2504, 15977, or 14760 nucleic acid, or the 2504, 15977, or 14760 polypeptide after treatment, relative to the level before treatment, is indicative of the efficacy of the treatment of the disorder.
In yet another aspect, the invention features a method for identifying an agent, e.g., a compound, which modulates the activity or expression of a 2504, 15977, and 14760 polypeptide, e.g., a 2504, 15977, and 14760 polypeptide as described herein, or a 2504, 15977, and 14760 nucleic acid, e.g., a 2504, 15977, and 14760 nucleic acid as described herein. The method includes contacting the 2504, 15977, and 14760 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect of the test compound on the activity of the polypeptide or nucleic acid to thereby identify a compound which modulates the activity of the polypeptide or nucleic acid.
In a preferred embodiment, the activity of the 2504, 15977, and 14760 polypeptide is a protein kinase activity.
In a preferred embodiment, the activity of the 2504, 15977, and 14760 polypeptide is proliferation, differentiation, and/or survival of a cell, e.g., a 2504, 15977, and 14760-expressing cell, e.g., a neural cell, a cardiovascular cell, a hematopoietic cell (e.g., a bone marrow cell such as a glycophorin-positive cell, an erythroid cell, a megakaryocyte).
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or an 2504, 15977, and 14760 nucleic acid, or any combination thereof.
In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 2504, 15977, and 14760 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 2504, 15977, and 14760 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 2504, 15977, and 14760 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.